Apoptosis Assessment

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Chapter 9 Apoptosis Assessment

image Introduction

Apoptosis is a distinct form of cell death controlled by an internally encoded suicide program. It is believed to occur in the majority of animal cells. It is a distinct event that triggers characteristic morphologic and biological changes in the cellular life cycle. It is common during embryogenesis, normal tissue and organ involution, and cytotoxic immunologic reactions, and occurs naturally at the end of the life span of differentiated cells. Apoptosis can also be induced in cells by the application of a number of different agents, including physiologic activators, heat shock, bacterial toxins, oncogenes, chemotherapeutic drugs, various toxic chemicals, ultraviolet and γ-radiation, and hypoxia. When apoptosis occurs, the nucleus and cytoplasm of the cell often fragment into membrane-bound apoptotic bodies, which are then phagocytized by neighboring cells. Alternatively, during necrosis, cell death occurs by direct injury to cells, resulting in cellular lysing and release of cytoplasmic components into the surrounding environment, often inducing an inflammatory response in the tissue. Apoptosis may occur in one cell, leaving surrounding cells unaffected, as opposed to necrosis, which affects multiple cells simultaneously.

A landmark of cellular self-destruction by apoptosis is the activation of nucleases and proteases that degrade the higher order chromatin structure of the DNA into fragments of 50 to 300 kilobases and subsequently into smaller DNA pieces of about 200 base-pairs in length. Activation of proteases, notably aspartate-specific cysteinyl proteases, referred to as caspases, is of primary relevance to apoptosis. Caspase-3 is considered to be the key mediator of apoptosis of mammalian cells, and its expression may be measured with immunohistochemical staining. Using fluorescent-labeled reagents, it is possible to tag the DNA break and identify the percentage of apoptotic cells with a high degree of accuracy.16

Measurable Features of Apoptosis

One of the most easily measured features of apoptotic cells is the breakup of the genomic DNA by cellular nucleases. These DNA fragments can be extracted from apoptotic cells and result in the appearance of DNA laddering when the DNA is analyzed by agarose gel electrophoresis. The DNA of nonapoptotic cells, which remains largely intact, does not display this laddering on agarose gels during electrophoresis. The large number of DNA fragments appearing in apoptotic cells results in a multitude of 3’-hydroxyl termini of DNA ends. This property can also be used to identify apoptotic cells by labeling the DNA breaks with fluorescent-tagged deoxyuridine triphosphate nucleotides. The enzyme terminal deoxynucleotidyl transferase catalyzes a template-independent addition of deoxyribonucleotide triphosphates to the 3’-hydroxyl ends of double- or single-stranded DNA. A substantial number of these sites are available in apoptotic cells, providing the basis for the single-step fluorescent labeling and flow cytometric method. Nonapoptotic cells do not incorporate significant amounts of the fluorescent-tagged deoxyuridine triphosphate nucleotides due to the lack of exposed 3’-hydroxyl DNA ends.

Apoptosis can also be characterized by changes in cell membrane structure. During apoptosis, the cell membrane’s phospholipid asymmetry changes—phosphatidylserine is exposed on the outer membrane, whereas membrane integrity is maintained. Annexin V specifically binds phosphatidylserine, whereas propidium iodide is a DNA-binding fluorochrome. When a cell population is exposed to both reagents, apoptotic cells stain positive for annexin V and negative for propidium iodide; necrotic cells stain positive for both, and live cells stain negative for both.3

This process of apoptosis and its analysis by flow cytometry are shown in Figures 9-1 and 9-2.

Another assessment of apoptosis involves ex vivo cell analysis. Specifically, the expression of active caspase-3 along with the Bcl-2/Bax ratio as markers of apoptosis can be measured. Immunohistochemical staining will reveal the expression of these apoptotic-related proteins, caspase-3 and cleaved caspase-3; the latter is indicative of apoptosis.7 Bcl-2 is anti-apoptotic gene product that exists in ratio to Bax and Bak, which are pro-apoptotic gene products. This ratio is indicative of the degree of apoptosis, with a decreased Bcl-2:Bax ratio indicative of apoptosis. Cells from Bax (−/−) and Bak(−/−) knockout animals do not respond to apoptosis inducers. In these cells, cytochrome C is not released from the mitochondrial membrane to initiate the caspase cascade.8 Thus, Bax and Bak are critical to apoptosis, and their expression in relation to Bcl-2 is highly correlative to apoptosis.

Apoptosis is Induced by Chemicals to Control Malignancy

Many chemicals have the capacity to bind to DNA, form DNA adducts, or cause DNA single-strand breaks, possibly leading to cancer. However, the body is equipped with many factors, enzymes, suppressor genes, and cellular sensors, all with the capacity to prevent the consequences of this DNA damage by activating apoptosis-inducing signals.

The role of apoptosis in regulating tissue growth is readily apparent in the simple equation in which the rate of growth is equal to the difference between the rates of cell proliferation and cell death. Thus, tissues expand if the rate of proliferation exceeds the rate of cell death. This is one of the reasons for suggesting that defects in apoptosis may contribute to the transformed state.

An important prediction of the relevance of apoptosis to malignancy is that the rate of apoptosis versus mitosis should influence the behavior of a tumor. Recently, the relationship between the apoptotic and mitotic indexes in a tumor was demonstrated as predictive of outcome: a higher ratio of apoptosis to mitosis within the tumor correlated with positive prognosis. Further, it was found that this was not simply a function of cell death per se. Tumors with a high incidence of necrosis rather than apoptosis were correlated with poor prognosis. It therefore follows that treatments or conditions that favor apoptosis should have desirable effects, and that defects in the pathways leading to apoptosis are likely to play important roles in the process of oncogenesis.4,5

Many reactive chemicals and drugs such as acetaminophen, diquat, carbon tetrachloride, quinones, cyanide, polyhydroxyl polyether, methyl mercury, and organotin have been implicated in apoptosis (programmed cell death) and necrosis (toxic cell death).916

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